Phagotosis by macrophages is an essential component of innate immunity. Although post-phagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis and evade macrophage defense mechanisms. Listeria monocytogenes is an intracellular pathogen that survives by passing from phagosomes into cytoplasm. Activation of macrophages with interferon-g plus LPS or TNFa increases resistance to many pathogens, including L. monocytogenes. The long-term objective of these studies is to identify those features of macrophage endocytic compartments that counteract intracellular pathogens. The hypothesis to be investigated in this proposal is that increased resistance to pathogens in activated macrophages results from altered phagosome progression to lysosomes, plus localized delivery of toxic compounds into acidic, late endosome-like phagosomes. To test this, quantitative fluorometric methods will be used to measure endocytic compartment dynamics and physiology in activated and non-activated macrphages. The first specific aim is to measure rates of phagosome maturation, fusion and lysosomes, and fluid-phase solute recycling. In the second aim, biochemical and fluorescence microscopic methods will be used to measure intravacuolar pH and intracellular nitric oxide levels. Sites of peroxynitrite generation will be localized in the cytopolasm and in individual phagosomes. The third specific aim is to identify host and bacterial factors that influence escape of L. monocytogenes from phagosomes. L. monocytogenes secretes a hemolytic protein, listeriolysin O ( LLO ), which mediates bacterial passage from phagosomes to cytoplasm. Fluorescence microscopy will be used to identify the compartment permeabilized by L. monocytogenes , and to determine how this compartment is altered in activated macrophages. Features of LLO that mediate escape from phagosomes will be identified using bacteria expressing mutant and wild-type LLO. For each mutant, the compartment of escape, the efficiency of perforation and escape, and the pH of perforation will be determined. Both listericidal and nonlistericidal macrophages will be compared, as well as macrophages from mice with induced deletions for components of the nitric oxide or superoxide biosynthetic pathways. Because these studies will provide direct measurements of conditions inside the vacuolar compartments of activated macrophages, the results should improve understanding of host defense mechanism related to infecton by L. monocytogenes as well as other intracellular pathogens. Response to The Previous Summary Statement This is a revised application to continue studies on identifying features of the macrophage vacuolar compartments that counteract intracellular pathogens with particular emphasis on Listeria monocytogenes. Several serious criticisms were directed at the earlier proposal. Most importantly, it was felt that the research plan was underdeveloped in that it contained too limited a range of experimental approaches. Additional criticisms were that the specificity of the probes for nitric oxide ( NO ) were not clear and the methods for measuring localized reactive oxygen species ( ROS ) and NO were poorly developed. Similarly, the rationale for why decreased rates of endocytic delivery to lysosomes should enhance microbicidal activity was not obvious and required further explanation. It was also felt by one reviewer that the PI should place greater emphasis on how listericidal macrophages inhibit listeriolysin LLO function. The PI has now responded to all of these criticisms/concerns in a direct, straightforward manner. In particular, the experimental plan has been expanded to include biochemical and immunohistochemical studies to buttress the morphologic experiments. The PI has provided evidence that the probes for NO are highly selective and expanded the methods for monitoring ROS and NO production. The description of these techniques and the necessary controls are now very well developed. Similarly, the "Background and Significance" section has been rewritten and provides a much better explanation of how alterations in membrane trafficking can favor the generation of reactive oxygen and nitrogen species. Specific Aim #3 now contains detailed experiments for investigating the chemical reactions of activated macrophages that may inhibit the hemolytic activity of LLO. The new sections utilizing biochemical methods to monitor NO formation, protein nitrosylation and evaluating the effects of NO and ROS on isolated LLO and Listeria in vitro are state of the art and highly relevant to this proposal. In short, the PI has successfully addressed all of the earlier criticism. These changes have resulted in a more focused, stronger proposal. Progress During the Past Funding Period The PI successfully accomplished the research described in the specific aims of his last funded proposal. For example, he demonstrated that the chemistry of particles undergoing phagocytosis can affect the degree of post-lysosomal interactions with other organelles, that increasing vacuolar pH reduced/prevented perforation of macrophages by Listeria monocytogenes, and that the formation of "spacious phagosomes" contributes to the survival and virulence of Salmonella. The PI also developed a highly efficient method for delivering macromolecules into the cytosol of macrophages using liposomes that contain LLO. This research resulted in ca. 10 papers published in first rate, peer-reviewed journals along with numerous reviews and book chapters. The PI now wishes to build on this strong record and extend these studies.